1. Field of the Invention
The present invention relates to the field of thrombotic disorders and diagnostic tests to predict their onset. More specifically, the present invention relates to the field of early diagnostic tests for the prediction of thrombotic events such as myocardial infarction, thromboembolism, stroke and related conditions.
In that the method whereby these thrombotic events are predicted is based on the presence of thrombospondin on the surface of a very small number of activated cells and more importantly a much larger number of resting platelets, the present invention relates to methods of analyzing resting platelets and their associated molecules. More specifically, the present invention relates to methods of analyzing resting platelets to qualitatively and quantitatively measure thrombospondin present on TSP receptors on the surface of resting platelets in a biological sample. Most specifically, the present invention relates to the field of determining thrombospondin concentrations as present on the population of resting platelets in a biological sample.
The invention also relates to the field of monoclonal and polyclonal antibodies, more particularly, a specific monoclonal antibody capable of immunologically binding with thrombospondin present on the surface of resting platelet surface TSP receptors is most preferably used in the described diagnostic and predictive methods.
As the present invention relates generally to the field of method for monitoring slight increases in platelet activation the described invention also relates to methods by which the effects of anti-thrombotic regimens in post-thrombotic patients can be evaluated and monitored in the patient. The present invention also relates to the field of post-thrombotic event monitoring systems, as the present techniques can also be used to determine if post-thrombotic resting platelet TSP concentrations have returned to "normal" or pre-thrombotic levels.
The present invention also relates to the field of diagnostic kits, as a particular diagnostic kit for use in the screening of biological samples to determine persons at risk of a thrombotic event is disclosed. The prediction of a thrombotic event is accomplished by the inclusion in the diagnostic kit of a monoclonal or polyclonal antibody (or Fab fragment thereof) which is capable of binding thrombospondin on resting platelets. In the disclosed diagnostic test, elevated levels of antibody binding to resting platelets indicate a person at potential risk of a thrombotic event.
2. Description of the Related Art
Cardiovascular disease presents a serious health risk throughout the world and is the leading cause of death in the United States. According to the American Heart Association, as of 1988, 6.08 million Americans suffered from coronary disease, while as of this year an additional 2.93 million Americans suffered from the devastating after effects of stroke. During 1988, a less fortunate 150,300 Americans died of stroke, while 511,050 died of coronary heart failure. More than 300,000 people a year die of heart attack prior to reaching a medical facility..sup.36 These statistics clearly support the need for a clinical test to identify these individuals before a major thrombotic event occurs.
In addition to these distressing statistics, the estimated cost of cardiovascular diseases in 1991 in terms of health care and medication in the United States alone was estimated to be about 101.3 billion dollars. An itemized analysis of these costs reveals that only 5.4 billion dollars (less than 6% of the total estimated expenditures) was related to the cost of medications in treating these conditions, while the remainder relates to costs associated with hospitalization, nursing care and lost output..sup.36 Clearly, these figures reflect that the current clinical management of these conditions is through the treatment of post-event symptoms, rather than through regimens to prevent or reduce the severity of an oncoming thrombotic event.
One reason for the lack of emphasis on clinical techniques for the prevention of a thrombotic event is the clinical inadequacy of current methods to timely indicate an oncoming thrombotic event. Generally, there are no symptoms preceding a damaging thrombotic event, such as a thrombogenic stroke. Some limited platelet activation has been reported to preclude myocardial infarction, thrombotic stroke, and deep venous thrombosis..sup.4, 5, 17, 33, 34, 36, 42, 47, 48 Therefore, several techniques proposed for detecting platelet activation have included the monitoring of platelet-secreted proteins, such as the several platelet proteins.47, 42, 4, 49, 50
Methods for examining the thrombotic event have been developed based on particular "marker" proteins, most specifically, proteins present on the surface of activated platelets..sup.47, 42, 4, 49, so However, the opportunity to clinically intervene in time to preclude thrombus formation is often lost by the time the concentration of activated platelets in the circulation reaches detectable levels.
An analysis of particular platelet proteins contemplated as potential "markers" of platelet activation and/or pre-indicators of thrombotic events requires their individual evaluation in light of the following criteria:
1. Clearance Time--The compound should not be rapidly cleared from the system being monitored. PA0 2. Measurability--The compound should be accurately measurable. PA0 3. Specificity--The compound must be specific for indicating an imminent increase in platelet activation, and hence onset of thrombus formation is soon to occur in vivo. PA0 The University of Texas Health Center at Tyler PA0 Department of Biochemistry PA0 P.O. Box 2003 PA0 Tyler, Texas 75710 PA0 The American Type Culture Collection PA0 12301 Parklawn Drive PA0 Rockville, Maryland 20852 PA0 TSP=thrombospondin PA0 Fg=fibrinogen PA0 Fn=fibronectin PA0 vWF=von Willebrand factor PA0 Kd=dissociation constant PA0 M=nanomolar PA0 .beta.TG=Beta-thromboglobulin PA0 Ca=calcium ion PA0 Mg=magnesium ion PA0 ADP=adenosinediphosphate PA0 EDTA=ethylenediamine tetraethylacetate PA0 PPACK=D-phenylalanyl-L-prolyl-L-arginine-chloromethylketone PA0 .mu.g=microgram PA0 .mu.l=microliter PA0 PGEI=prostaglandin E1 PA0 ACD=acid citrate dextrose PA0 BSA=bovine serum albumin PA0 IgG=immunoglobulin G PA0 ml=milliliter
Once platelets become activated, they do not enjoy a normal intravascular survival. The decreased intravascular survival of activated platelets tends to also decrease the sensitivity of an assay directed at the circulating activated platelets themselves..sup.57, 58
Several of the proposed platelet "marker" proteins are released as a consequence of the particular condition of the patient (diseased vs non-diseased state) and the particular handling and sampling techniques used in obtaining the sample..sup.47, 42, 4, 49, 50 For example, .beta.TG and PF4 are two platelet proteins which are released as a consequence of limited platelet activation, but these molecules are rapidly cleared from the circulation and a good correlation between elevated levels of these proteins in plasma and pre-thrombotic conditions has not been established..sup.47, 42, 4, 49, 50
Other adhesive platelet proteins, such as fibrinogen,.sup.37, 38 von Willebrand factor and fibronectin, bind only to GPIIb/IIIa complex present on the surface of physiologically "activated" platelets..sup.8, 59, 6, 64, 24, 60, 61, 62, 63, 65, 66
Antibodies directed against activated platelet secreted proteins are typically used to detect surface proteins via radioimmunoassay. Expression of these and other adhesive proteins on the platelet cell surface occurs upon platelet stimulation and secretion of alpha granule components. By way of example, particular proteins which are secreted by activated platelets include fibrinogen, fibronectin, von Willebrand factor, beta-thromboglobin (.beta.TG), platelet factor 4 and thrombospondin..sup.17, 33, 34, 36, 47, 42, 5, 48, 4, 49, 50, 8, 59, 6, 64, 24, 60, 61, 62, 63, 65, 66 Some of the secreted proteins appear on the surfaces of the small number of activated platelets while others are rapidly cleared from the circulation. Of these proteins, only thrombospondin is known to interact with not only the limited number of activated cells (which are likely to be cleared from the circulation by adhering to the endothelium), but also the larger population of circulating non-activated platelets..sup.67, 68, 69, 70, 71
While the GP11b/IIIb complex is present on both resting and activated platelets, these two components form a functional (i.e., able to support the binding of adhesive proteins to the platelet surface) "binding" site only when the platelet is in an activated state..sup.72, 73 Earlier studies have not been successful in detecting this very limited number of cells prior to thrombosis. A further deterrent to the measurement of activated platelets is due to the adhesive properties of activated cells which result in their rapid clearance from the circulation due to platelet aggregation and interaction with damaged endothelium..sup.57, 58 Thus, use of these particular platelet proteins as "markers" or "predictors" of thrombosis is not sufficiently sensitive enough to signal slight increases in platelet activation, or the beginnings of an imminent thrombotic event.
Although the degree of platelet adhesiveness has been correlated to the degree of thrombotic risk (Shaw, 1967), or with the incidence of thrombotic disease (Bygdeman, 1969), it has not been possible to predict whether an individual falls into a particular "high" risk group through monitoring platelet "adhesive" proteins. Primarily, the lack of sensitivity of the above "marker" proteins which are found only in association with the small population of activated platelets (due to the reasons cited above) make such measurements unreliable. In addition, such measurements are subject to considerable variability..sup.13
The Ginsberg patent describes a method for detecting activated platelets using an imaging reagent monoclonal antibody capable of binding thrombospondin expressed on the surface of activated platelets..sup.22 Specifically, the Ginsberg patent focuses on the detection of relatively large thrombi consisting of platelet aggregates within the body after the intravenous introduction of the radioactive imaging reagent monoclonal antibody into the patient. Clearly such a relatively invasive method, while of value in detecting the location of large thrombi in post-thrombotic patients, would have little value as a convenient non-invasive, predictive methodology. The relatively low resolution and sensitivity of the imaging techniques employed would preclude detection of thrombospondin on the circulating population of resting platelets as well as small, nascent platelet aggregates that would be expected in pre-thrombotic patients. Currently, the inability to detect platelet activation early in the pre-thrombotic stage often results in the loss of an opportunity to effectively intervene clinically to halt exponential platelet activation and thrombi formation.
Assay methods used to monitor activated platelets include standard radioimmunoassay for proteins released into the plasma by activated platelets and flow cytometric immunological detection of activated platelets utilizing fluorescently labeled antibodies which interact specifically with the activated cell population..sup.17, 33, 34 ,36, 47, 42, 5, 48, 4, 49, 50, 8, 59, 6, 64, 24, 60, 61, 62, 63, 65, 66
Moreover, methods which rely on the analysis of activated platelets suffer from a lack of sensitivity due to the removal of activated platelets from the circulation. Activated platelets have adhesive qualities which mediate interactions with damaged endothelium and atherosclerotic plaques..sup.57, 58 As an example, a recent study attempted to detect activated platelets in blood samples from nine patients undergoing cardiopulmonary bypass surgery..sup.29 This study employed flow cytometry. Three antibodies specific for activated platelets were used. One antibody was specific for fibrinogen associated with the activated platelet surface. This antibody gave positive results with only 2 of the 9 patients. The second antibody recognized GMP-140 which becomes expressed on the surface of activated platelets. This antibody gave positive results for only 1 of the 9 patients. A third antibody (PAC-1) directed against the active form of the fibrinogen receptor, GPIIb-IIIa, was positive in 5 out of 9 patients. It was also reported that the interaction of the anti-GMP-140 (S12) with activated platelets decreased when the cells were fixed with paraformaldehyde. This was also true for the PAC-1 antibody.
The above complication required the samples to be analyzed immediately to avoid spontaneous activation of the platelets which would invalidate the results. This would be cumbersome at best and most likely impossible in a clinical setting. Even if the samples could be processed quickly enough, the lack of sensitivity even under the most favorable conditions would make these procedures useless in detecting limited platelet activation which occurs prior to a thrombotic event.
In addition, these investigators measured the increase in platelet-derived microparticles and found a 2-fold increase, but could not definitely correlate this observation with increased platelet activation due to the surgery as opposed to the mechanical shear factors involved. As these blood samples were taken directly from an access port in the bypass circuit with little or no opportunity for the activated cells to be cleared (it is possible that some of the activated cells adhered to the bypass circuit itself), these results are not promising.
It is clear that the above methods are not adequate to detect platelet activation under extreme conditions, much less the limited activation that would precede a thrombotic event. In addition, methods which rely on the detection of proteins secreted into the circulation are equally unreliable. In large part, this unreliability is due to the rapid clearance rate of these proteins.sup.13. Another contributing factor is the large variability in the established normal range of concentrations of these proteins in the plasma..sup.13
Detection and measurement methods which in general depend on activated platelets are therefore unreliable because of: (1) endothelial removal of activated platelets; (2) lack of sufficient sensitivity and/or "signal-to-noise" ratio necessary to detect the small percentages of activated platelets that are present; (3) large variability in established normal ranges; and (4) in some procedures, the requirement to process samples immediately is unfeasible.
Thrombospondin is now known to be synthesized by a variety of cells including megakaryocytes, endothelial cells, smooth muscle cells, fibroblasts, monocytes and macrophages..sup.6, 8 Thrombospondin resides primarily in endogenous pools within the alpha-granules of platelets, and unlike other platelet proteins, is present in very low concentrations in plasma (20-165 ng/ml in blood plasma)..sup.8
The capacity of thrombospondin to interact with resting cells makes it unique among the platelet adhesive glycoproteins..sup.8 In comparison to other platelet proteins, the concentration of thrombospondin has been reported to be 3 to 5 orders of magnitude less than fibronectin or fibrinogen blood plasma levels..sup.8 The low levels of TSP associated with resting platelets is also consistent with detectable plasma levels of thrombospondin.
Thrombospondin is known to have functional surface receptors on both activated and resting platelets..sup.8 A substantial body of evidence suggests that TSP is either absent, or only present at minimal levels, on the surface of resting platelets..sup.6, 8 As already noted, blood platelets mobilize certain cytoplasmic storage granules (alpha granules) upon "activation". These alpha granules release thrombospondin into the extracellular environment upon platelet activation. TSP then binds TSP receptors located on activated platelet surfaces. Released thrombospondin also binds to non-stimulated or resting platelets at a separate population of high affinity TSP surface receptors..sup.43 The binding affinity of endogenous thrombospondin for resting platelets is particularly high, with a Kd less than 4 nM, the binding affinity between the activated platelet receptor and thrombospondin has been estimated at 250 nM..sup.7 At the normal plasma concentration of TSP, which has been estimated to be as high as 20 to 165 ng/ml.sup.8, it has been predicted that there may exist from 30 to 225 molecules of TSP on the surface of a resting platelet..sup.7, 8
A resting platelet contains approximately 3,000 high-affinity receptor sites on its surface at which TSP can bind..sup.3 Thus, the binding of thrombospondin to resting platelets is described as having a low-capacity mechanism, in that resting platelets are capable of binding a maximum of 3000 molecules of thrombospondin per resting platelet cell.
In contrast, an activated platelet contains approximately 36,000 low affinity receptor sites on its surface at which TSP can bind. Thus, the interaction of thrombin-stimulated platelets (i.e., "activated" platelets) is characterized as having a lower affinity (kd=250 nM) and high capacity..sup.8
In contrast, many other platelet proteins proposed as potential "markers" of thrombosis are normally present at very high concentrations in the circulation (e.g., fibrinogen). A high relative plasma concentration of a particular protein would make it difficult to detect small changes in that particular protein concentration. Additionally, fibrinogen is difficult to detect on the surface of platelets. Specifically, the number of platelets which interact with fibrinogen is small, making the detection of platelets with surface-expressed fibrinogen difficult, if not impossible. Other proteins (e.g., .beta.TG), while not normally present at high plasma concentrations, are cleared from the system, so rapidly that it becomes impossible to accurately relate elevated plasma levels with low level platelet activation..sup.74, 75, 76, 77, 78, 79
Thus, both rapidly cleared, or high plasma concentration platelet proteins are rendered unreliable as in vivo thrombotic event indicators.
Since platelet activation is a preliminary event to thrombosis, the probability of which exponentially increases following injury to blood vessels and soft tissues, it is important to develop methods by which the onset of early platelet activation may be accurately predicted, particularly at low "activated" platelet concentrations. A system which was capable of predicting the onset of early platelet activation would aid in the prediction of those patients at risk of a thrombotic disorder. Most preferably, a sensitive and specific method for identifying a "prethrombotic state" would be relatively independent of the particular concentration of activated platelets in the circulation. Such would allow early thrombus detection, prior to the beginning of exponential platelet activation, and thereby increase the probability of halting an oncoming thrombotic event.
Such a method would also be useful in post-thrombosis treatment evaluations to determine the effectiveness of prescribed therapy. Current methods of post-thrombosis treatment evaluation monitor only blood fibrinopeptide thrombus breakdown products. Several post-thrombus therapies act to inactivate (maintain in a resting state) the platelets, and typically reduce future thrombus formation triggered by limited platelet activation .sup.40 However, this method does not measure platelet activation directly, thus, it cannot determine if a specific regimen is effective enough to prevent future thrombus formation.
As thrombotic events, such as myocardial infarction, thrombotic stroke, and deep venous thrombosis, occur without preceding symptoms other than limited, relatively undetectable platelet activation, a particular need exists for the development of a non-invasive, rapid clinical method for reliably predicting the onset of thrombosis. Similarly, a need also exists for an in vivo screening method for detecting persons at risk for such thrombotic events so as to provide more clinically effective thrombosis-preventive therapies. This screening method would also be extremely useful for monitoring the effectiveness of post-thrombotic therapies.